Diabetic retinopathy represents one of the most debilitating microvascular complications of diabetes. It can lead to blindness in its final stage (Grange, 1995; Frank, 1996; Aiello L P et al., 1998). It is the second leading cause of acquired blindness in developed countries, after macular degeneration of the aged (Nathan et al., 1991). The risk of a diabetic patient becoming blind is estimated to be 25 times greater than that of the general population (Kahn and Hiller, 1974). At present there is no preventive or curative pharmacological treatment for this complication. The only treatment is laser retinal photocoagulation or vitrectomy in the most severe cases (Frank, 1995; Aiello, 1998).
Diabetic retinopathy is a progressive diabetic complication. It advances from a stage referred to as “simple” or initial (background retinopathy) to a final stage referred to as “proliferative retinopathy” in which there is formation of fragile retinal neovessels, leading to severe hemorrhages, sometimes with detachment of the retina, and to loss of vision (Grange, 1995; Frank, 1995). The microvascular lesions in simple retinopathy are characterized by microaneurysms, small petechial hemorrhages, exudates and venous dilations (Palmberg, 1977; ETDRS report no. 10, 1991). This simple retinopathy form can remain clinically silent for a long period of time. At this simple retinopathy stage cellular and structural deterioration of the retinal capillary can be observed in the postmortem examinations of retinas from diabetic patients, compared to the retinas from normal subjects of comparable age. As shown in FIG. 1, the lumens of the retinal capillaries are lined with endothelial cells. Pericytes (or mural cells) are located on the exterior and buried in the basal membrane of the vessel.
In the human retina and the retina of the rat, the numerical ratio of the pericytes to the endothelial cells is one to one (Kuwabara and Cogan, 1963). The alterations observed at this early stage consist of a thickening of the basal membrane of the capillaries (Friedenwald, 1950) and a selective disappearance of the pericytes (Cogan et al., 1961; Kuwabara and Cogan, 1963), diminishing the numerical ratio of the pericytes to the endothelial cells of the retinal capillaries to 0.3 to 1 in the pathological situation and even to 0.1 to 1 for the final stages (Cogan et al., 1961; Kuwabara and Cogan, 1963).
Studies performed on human retinas collected postmortem from patients with diabetes of long duration have shown that the pericytes die from apoptosis, programmed cell death, and not necrosis, the abrupt death seen subsequent to toxic attack (Mizutani et al., 1996; Li et al., 1997; Podesta et al., 2000). But the intracellular signalization pathway(s) by which they disappear is (are) not known.
Detection of apoptotic pericytes was performed in situ on intact retinas by a labeling technique for the nuclei of cells that passed into apoptosis, the TUNEL method (terminal deoxynucleotidyl transferase mediated dUDP nick-end labeling) (Mizutani et al., 1996). Supporting these data, certain pericytes that pass into apoptosis are also stained when use is made of an antibody directed against a protein involved in apoptosis named Bax (Podesta et al., 2000).
Another research team has demonstrated, after purification of the pericytes of retinas from diabetic patients, an augmentation in the expression of the gene of another protein involved in apoptosis, caspase 3 or CPP32 (Cysteine Protease Protein 32 kDa), by detecting an increased mRNA level of this protein by an inverse transcriptase chain polymerization amplification technique (Li et al., 1999). These purified pericytes also show augmented expression of other genes involved in the cellular antioxidant defenses such as that of, e.g., glutathione peroxidase, suggesting that oxidant stress could be involved in the disappearance by apoptosis of the retinal pericytes (Li et al., 1999).
Earlier studies carried out by The Diabetes Control Complications Trial Research Group (DCCT) (1993) and the UK Prospective Diabetes Study Group (UKPDS) (1998 a and b) already demonstrated the key role of the control of hyperglycemia in the development of diabetic retinopathy. Multiple studies determined that glucose, at pathological concentrations, could cause biochemical changes capable of altering the physiological functions or viability of the retinal microvascular cells. The inhibitor effect of glucose in vitro on the proliferation of pericytes, while the growth of endothelial cells is not modified (Porta et al., 1994) could, for example, explain the selective disappearance of pericytes over the course of retinopathy. Similarly, the synthesis of components of the basal membrane (collagen, laminin, fibronectin) stimulated in retinal pericytes and endothelial cells cultured in the presence of glucose (Li et al., 1984; Mandarino et al., 1993) could lead to the thickening of the basal membrane.
Another mechanism by which glucose could lead to vascular complications in diabetes is the increased production and accumulation of advanced products of glycation or AGE (Advanced Glycation End products) formed by the nonenzymic glycosylation or glycation of proteins, DNA or lipids (Maillard reaction, 1912) which have been demonstrated in numerous studies of diabetes (Thornalley, 1999). The amount of AGE measured in the skin of diabetic patients moreover correlates strongly with the severity of the vascular complications (Beisswenger et al., 1995). Glycation or the Maillard pathway shown in FIG. 2 describes the binding of reducing sugars to proteins: a reducing sugar in open form first reacts with the free amine group of basic amino acids contained in the proteins (lysine, arginine), leading to the formation of a Schiff base stabilized subsequently as an Amadori product.
These steps are reversible and dependent on the substrate concentrations (proteins and sugars). After formation of the Amadori product, it undergoes a series of modifications which lead either to an oxidative fragmentation and the formation of products of glycoxidation such as carboxymethyl lysine (CML), or to the formation of dicarbonyls such as 3-deoxyglucosone. These very reactive dicarbonyls in turn react with the protein amines, and thereby propagate the Maillard reaction, forming intramolecular and intermolecular bridges in long-lived proteins (Thornalley, 1999). Other AGE synthesis pathways in addition to glucose have been proposed recently.
Methylglyoxal, formed by the fragmentation of triose phosphates and the oxidation of acetone in the liver (by means of monooxygenases), is another dicarbonyl with an elevated blood concentration in subjects with diabetes (McLellan et al., 1994). In vitro, under physiological pH and temperature conditions, methylglyoxal can irreversibly modify especially the arginine residues of proteins (Lo et al., 1994). Furthermore, the AGE of proteins formed after reaction with methylglyoxal are described as major products observed in diabetes (Degenhardt et al., 1998). The formation of AGE of intracellular proteins after reaction with methylglyoxal, which is more reactive than glucose in the Maillard reaction, appears to be a dominant formation pathway in cells (Nishikawa et al., 2000, Shinohara et al., 1998).
In retinal microvessels, an accumulation of Amadori products, AGE precursors (Schalkwijk et al., 1999), as well as an accumulation of AGE was demonstrated using anti-AGE (or anti-Amadori) antibodies at the level of the basal membrane of pericytes and endothelial cells (Stitt et al., 1997). Multiple studies performed in vitro suggest that AGE could be involved in retinal vascular dysfunction. In fact, AGE modifies the proliferation of both cell types, inhibiting in particular the growth of pericytes (Chibber et al., 1997; Ruggiero-Lopez et al., 1997; Yamagishi et al., 1995). Extracellular AGE is capable of binding to specific membrane receptors and inducing various cellular responses. Studies have shown that there is a co-localization of AGE and AGE receptors in the retinal microvessels of diabetic rats (Stitt et al., 1997; Soulis et al., 1997). These receptors were identified on different cell types, including pericytes (Chibber et al., 1997; Yamagishi et al., 1995; Stitt et al., 1997; Thornalley, 1998) and different types of receptors were isolated: p60, p90 (Yang et al., 1991), RAGE (Receptor for AGE), which is a receptor of the immunoglobulin superfamily (Neeper et al., 1992) complexed to lactoferrin (Schmidt et al., 1992) and galectin 3 (Vlassara et al., 1995). RAGE, the best characterized of the receptors and which is present on the pericytes (Yamagishi et al., 1995; Brett et al., 1993) after having bound an AGE, induced an intracellular oxidant stress (Yan et al., 1994) and activation of the NF-KB transcription factor (Yan et al., 1994) as well as the activation of p21 ras and MAP (Mitogen Activation Protein) kinase (Lander et al., 1997) in the endothelial cells of the umbilical cord vein. Activation of the NF-KB transcription factor in these cells can be inhibited by various antioxidants such as lipoic acid (Bierhaus et al., 1997) and antioxidant enzymes (Yan et al., 1994). The antioxidant trilox can prevent the loss of pericytes in the retina of diabetic rats (Ansari et al., 1998), suggesting that oxidant stress could be involved in the disappearance of pericytes. An antisense RNA directed against the RAGE receptor corrects the AGE antiproliferative effect observed on the pericytes (Yamagishi et al., 1995).
The importance of the Maillard pathway and the accumulation of AGE in the pathogenesis of diabetic complications, and of retinopathy in particular, has been explored notably by using glycation inhibitors such as aminoguanidine, which by its free amine traps the reactive glucose or dicarbonyls, preventing them from reacting with the proteins. In a streptozotocin diabetic rat model, aminoguanidine prevents the accumulation of AGE in the retinal microvessels as well as the disappearance of pericytes and reduces by 80% the number of acellular capillaries in the retina (Hammes et al., 1991).
In contrast, the mechanisms by which AGE brings about the disappearance of pericytes as well as the mode of disappearance—apoptosis or necrosis—of pericytes due to AGE remain questions that are still unresolved.